Synopsis

The aim of this study was to determine whether diffusion behavior of metabolites in the congenital portal systemic shunt(PSS) mouse brain is different from ones of the healthy controls in vivo, combining large diffusion weighting and ¹H MRS methods. The diffusion behavior of most investigated metabolites except taurine was in excellent agreement with those in control mouse brain. The reduced diffusivities of taurine in PSS mice compared to control ones may be due of possible cellular redistribution of taurine in response to some osmotic perturbations in PSS mice in vivo, however, it needs to be further explored.

Purpose

Diffusion-weighted ¹H MRS allows investigating the cellular compartmentalization of molecules in the living organs, e.g. brain¹, and may shed insight on alterations of cellular restrictions faced by metabolites in different cerebral abnormalities and diseases. The neurochemical profile of congenital portal systemic shunt(PSS) mice displayed the abnormal neurochemical profile, consisting elevated glutamine(Gln) concentrations and reduced myo-inositol(Ins) concentrations², both of which are highly located in astrocytes. Thus, we hypothesized that diffusion behavior of metabolites in PSS mouse brain might be altered compared to their control. Therefore, the aim of this study was to determine whether diffusion behavior of metabolites in the PSS mouse brain is different from ones of the healthy mouse in vivo, combining large diffusion weighting and ¹H MRS methods.

Methods

All experimental procedures involving animals were approved by the local veterinary authorities. All experiments were performed on a 14.1T/26cm horizontal magnet with a 12-cm gradient coil insert (400mT/m, 120μs) using a home-built proton quadrature transceiver. Six adult PSS and six age-matched healthy(Ctrl) C57BL/6J mice (male, 30-35g) have been prepared and anesthetized using 1-15.% isoflurane. ¹H-MRS data were acquired using localized diffusion-weighed STEAM-based spectroscopic pulse sequence³ with optimum parameters(TE=16 ms and a mixing time of 113 ms¹). Diffusion weighting was applied simultaneously along three orthogonal diffusion gradient directions (i.e. x, y and z) to cover the wide b range of 0 to 45 ms/ μm². In vivo data was acquired with gradient duration of δ=6 ms, gradient separation of Δ=122 ms and gradient strength of 1, 13, 26, 52, 92, 143, 199 mT/m in a voxel of 91 μl (Fig 1) enabling sufficient sensitivity to correct the phase of single scan metabolite spectra at all b-values (Fig 2). Metabolite concentrations were quantified with LCModel. Data from the entire range of b-values in Fig 3 were fitted using following bi-exponential equation where ($$$D_{App}^{fast}$$$,$$$D_{App}^{slow}$$$) represent apparent diffusion coefficients of the fast and slow decaying signal components of each metabolite and $$$P_{App}^{slow}$$$ reflects the relative contribution of the slow component in the metabolite signal. S and S₀ are signal intensity with and without diffusion gradients. $$\frac{S}{S_{0}}=P_{App}^{slow}.exp(-b.D_{App}^{slow})+(1-P_{App}^{slow}).exp(-b.D_{App}^{fast})$$

Results

The remarkable sensitivity and spectral resolution of localized short-echo ¹H MRS at 14T allowed a precise measurement of the diffusion properties of metabolites, e.g. N-actyle-aspartate(NAA), glutamate(Glu), total creatine(Cr+PCr), taurine(Tau) and Ins in the brain of PSS and Ctrl mouse in vivo at very high diffusion weighting(Fig 2). With such extensive diffusion weighting range, i.e. from 0 to 45 ms/μm², a clear deviation from mono-exponential attenuation was observed for all investigated metabolites for both groups (Fig 3). $$$D_{App}^{fast}$$$ values of all investigated metabolites fell in the same range for both PSS and Ctrl groups(Table 1). Among all investigated metabolites, NAA showed the lowest $$$D_{App}^{slow}$$$. The elevated Gln levels in PSS mice allowed us to measure Gln diffusivity in the mouse brain in vivo. The $$$D_{App}^{fast}$$$ of Gln in PSS mouse brain was in the same range of other investigated metabolites. However, the $$$D_{App}^{slow}$$$ of Gln was higher than NAA(t-test, P<0.05), very similar to Ins and Cr+PCr(Table 1). The $$$D_{App}^{fast}$$$ and $$$D_{App}^{slow}$$$ values for most investigated metabolites(Table 1) were in excellent agreement with those in Ctrl mouse brain except that Tau showed slightly lowered $$$D_{App}^{slow}$$$ and $$$P_{App}^{slow}$$$ in PSS mice(t-test, p<0.05).

Discussion

A slight variation in $$$D_{App}^{slow}$$$ values between different metabolites suggested different intracellular distribution spaces for metabolites in the brain which was consistent with the proposed hypothesis about different localization of metabolites in the brain, such as NAA mostly in neurons⁴ and Ins mostly in astrocytes⁵. The diffusion behavior of Gln, similar to Ins but different from NAA, was in excellent agreement with the assumption made about considering a large pool of glutamine in astrocyte in ¹³C studies^{6, 7}. The comparable diffusion properties of most investigated metabolites in the brain in vivo between PSS and Ctrl mice may indicate that unaltered barrier and cellular restriction dominate on the diffusion of metabolites in both group and therefore could support the hypothesis about the similarity of intracellular distribution space for these metabolites in PSS mice when compared to Ctrl mice. The reduced diffusivities of Tau in PSS mice compared to Ctrl ones may be due of possible cellular redistribution of Tau in response to some osmotic perturbations in PSS mice in vivo⁸,however, it needs to be further explored.

Conclusion

Acknowledgements

References

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